Effects of Regulating Hippo and Wnt on the Development and Fate Differentiation of Bovine Embryo.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

Effects of ß-Nicotinamide Mononucleotide, Berberine, and Cordycepin on Lipid Droplet Content and Developmental Ability of Vitrified Bovine Oocytes.

Oocyte vitrification is crucial for livestock reproduction, germplasm conservation, and human-assisted reproduction, but the overabundance of lipids is highly detrimental to oocyte development. It is necessary to reduce the lipid droplet content of oocytes before cryopreservation. This study analyzed the impact of ß-nicotinamide mononucleotide (NMN), berberine (BER), or cordycepin (COR) on various aspects of bovine oocytes, including lipid droplet content and the expression levels of genes related to lipid synthesis in bovine oocytes, development ability, reactive oxygen species (ROS), apoptosis, and the expression levels of genes associated with endoplasmic reticulum (ER) stress, and mitochondrial function in vitrified bovine oocytes. The results of our study indicated that 1 µM NMN, 2.5 µM BER, and 1 µM COR were effective in reducing the lipid droplet content and suppressing the expression levels of genes involved in lipid synthesis in bovine oocytes. Our findings showed that the vitrified bovine oocytes treated with 1 µM of NMN had a significantly higher survival rate and better development ability compared to the other vitrified groups. Additionally, 1 µM NMN, 2.5 µM BER, and 1 µM COR decreased the levels of ROS and apoptosis, decreased the mRNA expression levels of genes involved in ER stress and mitochondrial fission but increased the mRNA expression levels of genes associated with mitochondrial fusion in the vitrified bovine oocytes. Our study results suggested that 1 µM NMN, 2.5 µM BER, and 1 µM COR effectively decreased the lipid droplet content and enhanced the development ability of vitrified bovine oocytes by lowering ROS levels, reducing ER stress, regulating mitochondrial function, and inhibiting apoptosis. Furthermore, the results showed that 1 µM NMN was more effective than 2.5 µM BER and 1 µM COR.

Combination of CNP, MT and FLI during IVM Significantly Improved the Quality and Development Abilities of Bovine Oocytes and IVF-Derived Embryos.

Oocyte maturation is a critical step in the completion of female gametogenesis in the ovary; thus, for subsequent fertilization and embryogenesis. Vitrification of embryo also has been shown to be closely associated with oocyte maturation. To improve the quality and developmental potential of bovine oocytes derived from in vitro maturation (IVM), Pre-IVM with C-type natriuretic peptide (CNP), melatonin (MT) and in combination, IGF1, FGF2, LIF (FLI) were supplemented in the IVM medium. In this current study, we cultured bovine oocytes in Pre-IVM with CNP for 6 h before transferring them to the IVM medium supplemented with MT and FLI. The developmental potential of bovine oocytes was then investigated by measuring the reactive oxygen species (ROS), the intracellular glutathione (GSH) and ATP levels, the transzonal projections (TZP), the mitochondrial membrane potential (ΔΨm), cacline-AM, and the expression of related genes (cumulus cells (CCs), oocytes, blastocysts). The results revealed that oocytes treated with a combination of CNP, MT, and FLI had dramatically improved the percentage of oocytes developed to blastocyst, ATP content, GSH levels, TZP intensity, the ΔΨm, cacline-AM fluorescence intensity, and considerably reduced ROS levels of oocytes. Furthermore, the survival rate and the hatched rate after vitrification of the CNP+MT+FLI group were significantly higher than those other groups. Thus, we speculated that CNP+MT+FLI increases the IVM of bovine oocytes. In conclusion, our findings deepen our understanding and provide new perspectives on targeting the combination of CNP, MT and FLI to enhance the quality and developmental potential of bovine oocytes.

A 1.1 Mb duplication CNV on chromosome 17 contributes to skeletal muscle development in Boer goats.

The Boer goat is one of the top meat breeds in modern animal husbandry and has attracted widespread attention for its unique growth performance. However, the genetic basis of muscle development in the Boer goat remains obscure. In this study, we identified specific structural variants in the Boer goat based on genome-wide selection signals and analyzed the basis of the molecular heredity of related candidate genes in muscle development. A total of 9 959 autosomal copy number variations (CNVs) were identified through selection signal analysis in 127 goat genomes. Specifically, we confirmed that the highest signal CNV (HSV) was a chromosomal arrangement containing an approximately 1.11 Mb (CHIR17: 60062304-61171840 bp) duplicated fragment inserted in reverse orientation and a 5 362 bp deleted region (CHIR17:60145940-60151302 bp) with overlapping genes (e.g., ARHGAP10, NR3C2, EDNRA, PRMT9, and TMEM184C). The homozygous duplicated HSV genotype (+/+) was found in 96% of Boer goats but was not detected in Eurasian goats and was only detected in 4% of indigenous African goats. The expression network of three candidate genes ( ARHGAP10, NR3C2, and EDNRA) regulating dose transcription was constructed by RNA sequencing. Results indicated that these genes were involved in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs) and their overexpression significantly increased the expression of SAA3. The HSV of the Boer goat contributed to superior skeletal muscle growth via the dose effects of overlapping genes.

Comparative analysis of the genetic diversity of the neutral microsatellite loci and second exon of the goat MHC-DQB1 gene.

This study compared and analyzed the genetic diversity and population structure of exon 2 of the DQB1 gene and 13 autosomal neutral microsatellite markers from 14 Chinese goat breeds to explore the potential evolutionary mechanism of the major histocompatibility complex (MHC). A total of 287 haplotypes were constructed from MHC-DQB1 exon 2 from 14 populations, and 82 nucleotide polymorphic sites (SNPs, 31.78%) and 172 heterozygous individuals (79.12%) were identified. The FST values of the microsatellites and MHC-DQB ranged between 0.01831-0.26907 and 0.00892-0.38871, respectively. Furthermore, 14 goat populations showed rich genetic diversity in the microsatellite loci and MHC-DQB1 exon 2. However, the population structure and phylogenetic relationship represented by the two markers were different. Positive selection and Tajima's D test results showed the occurrence of a diversified selection mechanism, which was primarily based on a positive and balancing selection in goat DQB. This study also found that the DQB sequences of bovines exhibited trans-species polymorphism (TSP) among species and families. In brief, this study indicated that positive and balancing selection played a major role in maintaining the genetic diversity of DQB, and TSP of MHC in bovines was common, which enhanced the understanding of the MHC evolution.

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 µg/ml) or (ii) CD9 antibody (1, 5 and 25 µg/ml) or (iii) CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

Genetic basis investigation of wattle phenotype in goat using genome-wide sequence data.

In domestic goats, wattles often appear in even numbers, mostly on the neck and a few under the ear. Goat wattle is composed of ectopic cartilage tissue covered by skin and was reported as a dominant inheritance. Thirty-eight goats from two Southwest Chinese breeds were studied to elucidate the genetic basis of wattle phenotype in goat. Their genomes were sequenced for wide-genome selective sweep analysis (WGSA) and a genome-wide association study (GWAS). The WGSA results revealed 500 candidate genes identified by fixation index and π ratio and 261 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched with 195 genes and 38 significantly enriched KEGG items. In particular, three chondrogenesis-related pathways (Wnt, Hippo and MAPK signaling pathways) were found. Among the 500 genes, 474 were enriched to 2855 Gene Ontology items, and four (BMP2, BMP4, RARA and MSX1) were annotated in the regulation and development of chondrogenesis. Four chondrogenesis-related genes (GREM1, NEDD4, ATG7 and ITGA1) were identified from 519 single-nucleotide polymorphisms (SNPs) with a GWAS above the threshold. Six and 11 SNPs on chromosome 10 are located on GREM1 and NEDD4 respectively, and the highest numbers of SNPs on chromosomes 20 and 22 are located on ITGA1 and ATG7 respectively. All of these genes are related to cartilage development. This study identified a series of genes related to chondroplasia by GWAS and WGSA and presented the possibility that wattle inheritance may be influenced by multiple genes. This work provides a new theoretical understanding of the hereditary basis of wattle phenotype.

Genome-Wide Selective Analysis of Boer Goat to Investigate the Dynamic Heredity Evolution under Different Stages.

Boer goats, as kemp in meat-type goats, are selected and bred from African indigenous goats under a long period of artificial selection. Their advantages in multiple economic traits, particularly their plump growth, have attracted worldwide attention. The current study displayed the genome-wide selection signature analyses of South African indigenous goat (AF), African Boer (BH), and Australian Boer (AS) to investigate the hereditary basis of artificial selection in different stages. Four methods (principal component analysis, nucleotide diversity, linkage disequilibrium decay, and neighbor-joining tree) implied the genomic diversity changes with different artificial selection intensities in Boer goats. In addition, the θπ, FST, and XP-CLR methods were used to search for the candidate signatures of positive selection in Boer goats. Consequently, 339 (BH vs. AF) and 295 (AS vs. BH) candidate genes were obtained from SNP data. Especially, 10 genes (e.g., BMPR1B, DNER, ITGAL, and KIT) under selection in both groups were identified. Functional annotation analysis revealed that these genes are potentially responsible for reproduction, metabolism, growth, and development. This study used genome-wide sequencing data to identify inheritance by artificial selection. The results of the current study are valuable for future molecular-assisted breeding and genetic improvement of goats.

Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-ß-Cyclodextrin Treatment.

Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-ß-cyclodextrin (CLC/MßCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MßCD.

Identification of differentially expressed microRNAs in ovulatory and subordinate follicles in Dazu black goats.

How multiple ovulations happen in prolific goats is still unknown even though studies on ovarian physiology and folliculogenesis have made extensive progress. MicroRNAs (miRNAs) are a class of endogenous regulatory factors and regulate structural gene expression mainly at the post-transcriptional level. In this study, in the follicular phase, six large follicles were collected from prolific Dazu black goat and used to generate RNA libraries for RNA sequencing. Based on the litter size and average number of ovulatory follicles in Dazu black goats, the largest three follicles were sorted as ovulatory follicles, and the remaining as subordinate ones. In total, 418 known miRNAs and 110 novel miRNAs were found, and the expression of six randomly selected miRNAs was validated by quantitative PCR analysis. Nine miRNAs were differently expressed between the ovulatory and subordinate follicles (p < 0.01). Chi-miR-582-5p, novel-130, chi-miR-214-3p, and chi-miR-500-5p were upregulated in the ovulatory group, and chi-miR-383, chi-miR-130b-5p, chi-miR-92a-3p, chi-miR-125b-5p, and novel-9 were downregulated in the same group. Chi-miR-130b-5p and chi-miR-214-3p were predicted to target at LHR (XM_013967581.1), GDF9 (NM_001285708.1), BMP15 (NM_001285588.1), and CYP19A1 (XM_013967046.1). In conclusion, nine miRNAs were differently expressed between ovulatory and subordinate follicles, and chi-miR-130b-5p and chi-miR-214-3p were predicted to regulate the expression of genes involved in gonadotropin hormone signaling and oocyte-derived growth factors. Additional studies are needed to confirm these findings.

Investigating genetic diversity and population phylogeny of five Chongqing local chicken populations autosomal using microsatellites.

The genetic diversity and population structures of five Chongqing local chicken populations were investigated using by 24 microsatellite markers. Results revealed that the mean number of alleles (NA) ranged from 7.08 (Daninghe chicken, DN) to 8.46 (Nanchuan chicken, NC). The highest observed heterozygosity (HO) and expected heterozygosity (HE) were observed in DN (HO = 0.7252; HE = 0.7409) and the lowest HO and HE were observed in XS (Xiushan native chicken [XS], HO = 0.5910 and HE = 0.6697). The inbreeding coefficient (FIS) within population ranged from 0.022 (DN) to 0.119 (XS). Among the 24 microsatellite markers, four loci (MCW0111, MCW0016, ADL0278, and MCW0104) deviated from the Hardy-Weinberg equilibrium in all the studied populations. The results of population polygenetic analysis based on Nei's genetic distance and STRUCTURE software showed that the clustering of the five populations was incomplete consistent with geographical distribution. Moreover, a large number of gene flows were widespread among different populations, suggesting that genetic material exchanges occurred due to human activities and migration which was also verified by PCoA. In summary, this study preliminarily showed that Chongqing local chicken populations had rich genetic diversity and remarkable genetic divergence, but still high risk in conversion. These findings would be useful to the management of conservation strategies and the utilization of local chicken populations in further.

Genome-wide selective detection of Mile red-bone goat using next-generation sequencing technology.

The ecotype population of goats (Capra hircus) was created by long-term artificial selection and natural adaptation. Mile red-bone goat is an indigenous breed with visible red bones, and its special bone structure has received extensive attention. This study aimed to identify genetic variants and candidate genes associated with specific bone phenotypes using next-generation sequencing technology (NGS). The results revealed that 31,828,206 single nucleotide polymorphisms (SNPs) were obtained from 72 goats (20 Mile red-bone goats and 52 common goats) by NGS. A total of 100 candidate genes were identified on the basis top 1% window interaction from nucleotide diversity (π), π ratio (π A/π B), and pairwise fixation index (F ST). Exactly 77 known signaling pathways were enriched. Specifically, three coding genes (NMNAT2, LOC102172983, and PNLIP) were annotated in the vitamin metabolism signaling pathways, and NCF2 was annotated to the osteoclast (OC) differentiation pathway. Furthermore, 5862 reliable copy number variations (CNVs) were obtained, and 14 and 24 genes were annotated with the top 1‰ CNV based on F ST (>0.490) and V ST (>0.527), respectively. Several pathways related to bone development and metabolism of exogenous substances in vivo, including calcium signaling pathway, OC differentiation, and glycerophospholipid metabolism, were annotated. Specifically, six genes from 19 candidate CNVs, which were obtained by interaction of the top 1‰ CNVs with F ST and V ST, were annotated to mucin-type O-glycan biosynthesis and metabolic pathways. Briefly, the results implied that pseudopurpurin and specific genetic variants work together to contribute to the red-bone color and specific bone structure of Mile red-bone goat. This study is helpful to understanding the genetic basis of the unique bone phenotype of Mile red-bone goats.

Genome-Wide Association Analysis of Growth Curve Parameters in Chinese Simmental Beef Cattle.

The objective of the present study was to perform a genome-wide association study (GWAS) for growth curve parameters using nonlinear models that fit original weight-age records. In this study, data from 808 Chinese Simmental beef cattle that were weighed at 0, 6, 12, and 18 months of age were used to fit the growth curve. The Gompertz model showed the highest coefficient of determination (R2 = 0.954). The parameters' mature body weight (A), time-scale parameter (b), and maturity rate (K) were treated as phenotypes for single-trait GWAS and multi-trait GWAS. In total, 9, 49, and 7 significant SNPs associated with A, b, and K were identified by single-trait GWAS; 22 significant single nucleotide polymorphisms (SNPs) were identified by multi-trait GWAS. Among them, we observed several candidate genes, including PLIN3, KCNS3, TMCO1, PRKAG3, ANGPTL2, IGF-1, SHISA9, and STK3, which were previously reported to associate with growth and development. Further research for these candidate genes may be useful for exploring the full genetic architecture underlying growth and development traits in livestock.

SNP of AHSA2 gene in three cattle breeds using snapshot technology.

Droughtmaster is a tropical breed of beef cattle that can survive in hot climates and easily adapt to torrid environments. These traits are important in livestock breeding. In this study, we genotyped five single-nucleotide polymorphisms (SNPs) of the AHSA2 gene from 190 cattle belonging to three different breeds (Droughtmaster, Angus and Simmental) by using snapshot technology. This work aimed to identify the valuable molecular marker of heat resistance in cattle. Results showed that Droughtmaster exhibited higher expected heterozygosity and polymorphic information content compared with the two other breeds. The AHSA2-1 locus deviated from the Hardy-Weinberg equilibrium in the Droughtmaster breed (P < 0.05). Two SNPs in Droughtmaster diverged significantly from Angus and Simmental. The SNPs were identified as AHSA2-3 and AHSA2-4, which were closely linked to the three breeds based on pair-wise FST. AHSA2-4 involved a missense mutation. In summary, the GG genotypes in AHSA2-3 and AHSA2-4 may be candidate genotypes associated with heat resistance traits and may serve as valuable genetic markers for breeding of heat-tolerant beef cattle in the future.

Evolutionary relationship and population structure of domestic Bovidae animals based on MHC-linked and neutral autosomal microsatellite markers.

Major histocompatibility complex (MHC) genes are critical for disease resistance or susceptibility responsible for host-pathogen interactions determined mainly by extensive polymorphisms in the MHC genes. Here, we examined the diversity and phylogenetic pattern of MHC haplotypes reconstructed using three MHC-linked microsatellite markers in 55 populations of five Bovidae species and compared them with those based on neutral autosomal microsatellite markers (NAMs). Three-hundred-and-forty MHC haplotypes were identified in 1453 Bovidae individuals, suggesting significantly higher polymorphism and heterozygosity compared with those based on NAMs. The ambitious boundaries in population differentiation (phylogenetic network, pairwise FST and STRUCTURE analyses) within and between species assessed using the MHC haplotypes were different from those revealed by NAMs associated closely with speciation, geographical distribution, domestication and management histories. In addition, the mean FST was significantly correlated negatively with the number of observed alleles (NA), observed (HO) and expected (HE) heterozygosity and polymorphism information content (PIC) (P < 0.05) in the MHC haplotype dataset while there was no correction of the mean FST estimates (P> 0.05) between the MHC haplotype and NAMs datasets. Analysis of molecular variance (AMOVA) revealed a lower percentage of total variance (PTV) between species/groups based on the MHC-linked microsatellites than NAMs. Therefore, it was inferred that individuals within populations accumulated as many MHC variants as possible to increase their heterozygosity and thus the survival rate of their affiliated populations and species, which eventually reduced population differentiation and thereby complicated their classification and phylogenetic relationship inference. In summary, host-pathogen coevolution and heterozygote advantage, rather than demographic history, act as key driving forces shaping the MHC diversity within the populations and determining the interspecific MHC diversity.

Genome-wide selective sweep analysis of the high-altitude adaptability of yaks by using the copy number variant.

The domestic yak (Bos grunniens) from the Qinghai-Tibet Plateau is an important animal model in high-altitude adaptation studies. Here, we performed the genome-wide selective sweep analysis to identify the candidate copy number variation (CNV) for the high-altitude adaptation of yaks. A total of 531 autosomal CNVs were determined from 29 yak genome-wide resequencing data (15 high- and 14 low-altitude distributions) by using a CNV caller with a CNV identification interval > 5 kb, CNV silhouette score > 0.7, and minimum allele frequency > 0.05. Most high-frequency CNVs were located at the exonic (44.63%) and intergenic (46.52%) regions. In accordance with the results of the selective sweep analysis, 7 candidate CNVs were identified from the interaction of the top 20 CNVs with highest divergence from the F ST and V ST between the low (LA) and high (HA) altitudes. Five genes (i.e., GRIK4, IFNLR1, LOC102275985, GRHL3, and LOC102275713) were also annotated from the seven candidate CNVs and their upstream and downstream ranges at 300 kb. GRIK4, IFNLR1, and LOC102275985 were enriched in five known signal pathways, namely, glutamatergic synapse, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and olfactory transduction. These pathways are involved in the environmental adaptability and various physiological functions of animals, especially the physiological regulation under a hypoxic environment. The results of this study advanced the understanding of CNV as an important genomic structure variant type that contributes to HA adaptation and helped further explain the molecular mechanisms underlying the altitude adaptability of yaks.

Identification of a Goat Intersexuality-Associated Novel Variant Through Genome-Wide Resequencing and Hi-C.

Background: Polled intersex syndrome (PIS) leads to reproductive disorders in goats and exerts a heavy influence on goat breeding. Since 2001, the core variant of an 11.7 kb deletion at ~129 Mb on chromosome 1 (CHI1) has been widely used as a genetic diagnostic criterion. In 2020, a ~0.48 Mb insertion within the PIS deletion was identified by sequencing in XX intersex goats. However, the suitability of this variation for the diagnosis of intersex goats worldwide and its further molecular genetic mechanism need to be clarified. Results: The whole-genome selective sweep of intersex goats from China was performed with whole-genome next-generation sequencing technology for large sample populations and a case-control study on interbreeds. A series of candidate genes related to the goat intersexuality phenotype were found. We further confirmed that a ~0.48 Mb duplicated fragment (including ERG and KCNJ15) downstream of the ~20 Mb PIS region was reversely inserted into the PIS locus in intersex Chinese goats and was consistent with that in European Saanen and Valais black-necked goats. High-throughput chromosome conformation capture (Hi-C) technology was then used to compare the 3D structures of the PIS variant neighborhood in CHI1 between intersex and non-intersex goats. A newly found structure was validated as an intrachromosomal rearrangement. This inserted duplication changed the original spatial structure of goat CHI1 and caused the appearance of several specific loop structures in the adjacent ~20 kb downstream region of FOXL2. Conclusions: Results suggested that the novel complex PIS variant genome was sufficient as a broad-spectrum clinical diagnostic marker of XX intersexuality in goats from Europe and China. A series of private dense loop structures caused by segment insertion into the PIS deletion might affect the expression of FOXL2 or other neighboring novel candidate genes. However, these structures require further in-depth molecular biological experimental verification. In general, this study provided new insights for future research on the molecular genetic mechanism underlying female-to-male sex reversal in goats.

Genome-wide analysis of Chongqing native intersexual goats using next-generation sequencing.

Sex reversal has been studied extensively in vertebrate species, particularly in domestic goats, because polled intersex syndrome (PIS) has seriously affected their production efficiency. In the present study, we used histopathologically diagnosed cases of PIS to identify correlated genomic regions and variants using representative selection signatures and performed GWAS using Restriction-Site Associated Resequencing DNA. We identified 171 single-nucleotide polymorphisms (SNPs) that may have contributed to this phenotype, and 53 SNPs were determined to be located in coding regions using a general linear model. The transcriptome data sets of differentially expressed genes (DEGs) in the pituitary tissues of intersexual and nonintersexual goats were examined using high-throughput technology. A total of 10,063 DEGs and 337 long noncoding RNAs were identified. The DEGs were clustered into 56 GO categories and determined to be significantly enriched in 53 signaling pathways by KEGG analysis. In addition, according to qPCR results, PSPO2 and FSH were significantly more highly expressed in sexually mature pituitary tissues of intersexual goats compared to healthy controls (nonintersexual). These results demonstrate that certain novel potential genomic regions may be responsible for intersexual goats, and the transcriptome data indicate that the regulation of various physiological systems is involved in intersexual goat development. Therefore, these results provide helpful data for understanding the molecular mechanisms of intersex syndrome in goats.